Modulating urea degradation in wine yeast

ABSTRACT

The invention provides yeast strains transformed to reduce nitrogen catabolite repression of a gene encoding a urea degrading enzymatic activity expressed by the yeast strain under fermenting conditions. Strains of  Saccharomyces cerevisiae  are for example provided having enhanced DUR1,2 urea carboxylase-allophanate hydrolase activity under wine fermenting conditions.

FIELD OF THE INVENTION

The invention is in the field of microbial biochemistry. In one aspect, the invention relates to the manipulation of biochemical pathways involving nitrogen catabolism in organisms capable of fermentation of carbohydrates to produce ethyl alcohol. In selected embodiments, the invention relates to cultures and processes for making wine and other products of fermentation.

BACKGROUND OF THE INVENTION

Arginine is one of the predominant amino acids present in grape musts (Henschke and Jiranek, 1993). Arginine is thought to be transported into the yeast cell by the general amino acid permease encoded by the GAP1 gene (Jauniaux and Grenson, 1990) or by the arginine permease encoded by the CAN1 gene (Hoffmann, 1985). In Saccharomyces cerevisiae, arginine is reportedly degraded into urea and ornithine by arginase, the product of the CAR1 gene (Middelhoven, 1964; Sumrada and Cooper, 1982).

The DUR1,2 gene encodes a bifunctional enzyme, urea carboxylase-allophanate hydrolase (Dur1,2; urea amidolyase) which can degrade urea to ammonia and CO₂. The urea carboxylase function is encoded separately in the enzyme from green algae, which catalyzes the reaction: ATP+urea+CO₂=ADP+phosphate+urea-1-carboxylate (EC 6.3.4.6; systematic name: urea:carbon-dioxide ligase (ADP-forming); other name(s): urease (ATP-hydrolysing); urea carboxylase (hydrolysing); ATP-urea amidolyase; CAS registry number: 9058-98-4; Roon et al., 1970; Roon and Levenberg, 1972; Sumrada and Cooper, 1982). The allophanate hydrolase function is also encoded separately in the enzyme from green algae, which catalyzes the reaction: urea-1-carboxylate+H₂O=2 CO₂+2 NH₃ (EC 3.5.1.54; systematic name: Urea-1-carboxylate amidohydrolase; Other name(s): allophanate lyase; CAS registry number: 79121-96-3; Maitz et al., 1982; Roon, et al., 1972; Sumrada and Cooper, 1982).

In S. cerevisiae, the DUR1,2 gene is subject to nitrogen catabolite repression (NCR) by preferred nitrogen sources present in grape must (Genbauffe and Cooper, 1991). Urea that is not degraded may be secreted by yeast cells into the fermenting grape must. Secreted urea can react with ethanol in the must to form ethyl carbamate, which has been shown to produce various benign and malignant tumours in a variety of experimental animals (Mirvisch, 1968) and may therefore be considered a potential health risk to humans.

SUMMARY OF THE INVENTION

In one aspect, the invention relates to the recognition that the concentration of urea in a fermenting grape must, wine or distilled beverage may be controlled by modulating the nitrogen catabolite repression of the DUR1,2 gene in S. cerevisiae encoding for urea carboxylase and allophanate hydrolase activities. In one aspect, the invention accordingly provides yeast strains transformed to reduce nitrogen catabolite repression of a gene encoding for urea degrading enzymatic activity expressed by the yeast strain under fermenting conditions. The yeast strain may for example be transformed with a recombinant nucleic acid comprising a coding sequence encoding the urea degrading enzymatic activity, or with a promoter adapted to mediate expression of the urea degrading enzymatic activity under fermenting conditions. In some embodiments, the invention uses native or modified sequences homologous to the S. cerevisiae DUR1,2 promoter and coding sequences. The yeast strains and nucleic acids of the invention may for example be used in process to produce fermented alcoholic beverages, such as wines and other products of fermentation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a graphic view of the upstream region of the DUR1,2 gene, ending at the start of the DUR1,2 coding sequence and runninng in the 3′ to 5′ direction (corresponding to SEQ ID NO: 3).The information in the Figure is derived from the published Saccharomyces cerevisiae chromosome II complete chromosome sequence (Genbank LOCUS NC_(—)001134, ACCESSION NC_(—)001134, REGION: complement (635670 . . . 643177), VERSION NC_(—)001134.2, GI:14270686; Feldmann, H., Aigle, M., Aljinovic, G., Andre, B., Baclet, M. C., Barthe, C., Baur, A., Becam, A. M., Biteau, N., Boles, E. et al. “Complete DNA sequence of yeast chromosome II” EMBO J. 13 (24), 5795-5809 (1994); Goffeau, A., Barrell, B. G., Bussey, H., Davis, R. W., Dujon, B., Feldmann, H., Galibert, F., Hoheisel, J. D., Jacq, C., Johnston, M., Louis, E. J., Mewes, H. W., Murakami, Y., Philippsen, P., Tettelin, H. and Oliver, S. G. “Life with 6000 genes” Science 274 (5287), 546 (1996)). The Figure also shows an encoded amino acid sequence running from the carboxy to amino direction (corresponding to SEQ ID No: 4).

FIG. 2 sets out the sequence of a portion of the upstream region of the DUR1,2 gene, ending at the DUR1,2 start codon ATG (SEQ ID No: 5). Two putative NCR element GATM(G) boxes are highlighted (one at position −54 to −58 and the other at position −320 to −324), as well as putative TATM boxes.

FIG. 3 shows up-regulation of DUR1,2 transcription and translation in yeast engineered with the DUR1,2 expression cassette. (A) DUR1,2 expression in transformed GVY400 cells containing (lane 2) or lacking (lane 1) the DUR1,2 cDNA were compared by Northern analysis of total RNA using α³²P-dATP labeled probe for DUR 1,2. HHFI encoding histone 4 was used as the RNA loading standard. (B) Urea amidolyase expression in transformed GVY400 cells containing (lane 3) or lacking (lane 2) DUR1,2 cDNA were visualized by a western blot of the biotinylated proteins present in total protein extracted from the transformants. Detection was by chemiluminescence onto X-ray film using streptavidin coupled to horseradish peroxidase. High molecular weight biotinylated standards (5 μg per lane) were included as molecular weight markers (lane 1).

FIG. 4 is a schematic illustration of the cloning strategy of the DUR1,2 gene into the pHVX2 plasmid to yield PJC1, according to the D1/TRISEC method (Dietmaler and Fabry, 1995). Genomic DNA obtained from S. cerevisiae TCY 1 was prepared according to standard procedures (Ausubel et al., 1995). The coding sequence of the DUR1,2 gene was amplified by PCR using the ExTaq (Takara) DNA polymerase. The primers used were ⁵TTMAMMTGACAGTTAGTTCCGATACA³ (SEQ ID NO: 1) for the 5′ end and; ⁵TCGAMAAGGTATTTCATGCCMTGTTATGAC³ SEQ ID NO: 2) for the 3′ end of the gene. The designated start codon and the complementary sequence to the stop codon are presented in boldface. The amplification product was treated with T4 DNA polymerase in order to remove some nucleotides on the 3′-flanking end. The 3′-5′ exonuclease activity of the enzyme was stopped when required by adding adequate nucleotides. Plasmid pHVX2 (Volschenk et al., 1997) was cut by EcoR1 and BgIII restriction enzymes and then treated with the Klenow fragment of the E. coli polymerase I. Restriction sites were partially filled in the presence of dATP and dGTP in order to have sequences compatible with the cloning of the insert.

FIG. 5 is a schematic illustration showing the cloning of the phleomycin resistance gene cassette into pJC1 (labeled pHVX2 or pRUR1,2) containing the DUR1,2 gene. Standard recombinant methods were employed to construct pJC2 (labeled pHVX2phleo or pDUR1,2phleo) (Ausubel et al., 1995).

FIG. 6 is a schematic illustration of the plasmid pJC2/DUR1,2phleo (labelled pDUR1,2phleo in the illustration), which is a multicopy, episomal S. cerevisiae-E. coli shuttle plasmid derived from the pJC2phleo vector, wherein the DUR1,2 gene is inserted between the regulatory sequences of the yeast phosphoglycerate kinase (PGK1) gene (promoter and terminator sequences). The LEU2 marker facilitates the selection of transformed yeast cells that are auxotrophic for leucine. The plasmid also contains the Tn5Ble gene driven by the constitutive TEF1 yeast promoter and CYC1 yeast terminator. Yeast cells carrying this cassette become resistant to phleomycin. The use of this positive selection marker may be particularly well suited to working with transformed industrial yeast strains that do not carry auxotrophic markers.

FIG. 7 is a graph showing the ability of transformed yeast of the invention to decrease the concentration of urea in a culture media. A haploid laboratory yeast strain interrupted at the DUR1,2 chromosomal locus was transformed with the pJC2/DUR1,2phleo plasmid or the pJC2phleo plasmid without the DUR1,2 gene. The use of this mutant strain allows the performance of the strain to be evaluated in the absence of background due to a urea amidolyase endogenous activity. Transformed cells were grown in minimal media containing 0.1% glutamine, harvested and used to inoculate fresh media. After 1 hour, 33 mg/L urea were added. Every 2 hours, aliquots of the culture were harvested, cells were broken open in the media with glass beads, and the cell debris removed by centrifugation. Once the supernatent was collected, enzymes were heat inactivated and the urea present in the sample measured. This method facilitates the measurement of intracellular and extracellular urea simultaneously, allowing a distinction to be made between the urea taken up by the cells and the urea metabolised by the cells. The abs600 nm was also regularly measured to check the growth stage of each culture. Standard deviation n=6. Data shown with solid circles is the urea concentration in media innoculated with the laboratory strain transformed with pJC2phleo. Data shown with solid squares is the urea concentration in the media innoculated with the laboratory strain transformed wtih pJC2/DUR1,2phleo. Data shown with empty circles is the abs600 nm of the pJC2phleo strain. Data shown with empty squares is the abs600 nm of the pJC2/DUR1,2phleo strain. The left Y axis is [urea] in mg/L. The right Y axis is abs600 nm. The X axis is hours after innoculation.

DETAILED DESCRIPTION OF THE INVENTION

In various aspects, the present invention relates to the modification of genes and the use of recombinant genes. In this context, the term “gene” is used in accordance with its usual definition, to mean an operatively linked group of nucleic acid sequences. The modification of a gene in the context of the present invention may include the modification of any one of the various sequences that are operatively linked in the gene. By “operatively linked” it is meant that the particular sequences interact either directly or indirectly to carry out their intended function, such as mediation or modulation of gene expression. The interaction of operatively linked sequences may for example be mediated by proteins that in turn interact with the sequences.

The expression of a gene will typically involve the creation of a polypeptide which is coded for by a portion of the gene. This process typically involves at least two steps: transcription of a coding sequence to form RNA, which may have a direct biological role itself or which may undergo translation of part of the mRNA into a polypeptide. Although the processes of transcription and translation are not fully understood, it is believed that the transcription of a DNA sequence into mRNA is controlled by several regions of DNA. Each region is a series of bases (i.e., a series of nucleotide residues comprising adenosine (A), thymidine Cr), cytidine (C), and guanidine (G)) which are in a desired sequence.

Regions which are usually present in a gene include a promoter sequence with a region that causes RNA polymerase and other transcription factors to associate with the promoter segment of DNA. The RNA polymerase normally travels along an intervening region of the promoter before initiating transcription at a transcription initiation sequence, that directs the RNA polymerase to begin synthesis of mRNA. The RNA polymerase is believed to begin the synthesis of mRNA an appropriate distance, such as about 20 to about 30 bases, beyond the transcription initiation sequence. The foregoing sequences are referred to collectively as the promoter region of the gene, which may include other elements that modify expression of the gene. Such complex promoters may contain one or more sequences which are involved in induction or repression of the gene.

In the context of the present invention, “promoter” means a nucleotide sequence capable of mediating or modulating transcription of a nucleotide sequence of interest in the desired spatial or temporal pattern and to the desired extent, when the transcriptional regulatory region is operably linked to the sequence of interest. A transcriptional regulatory region and a sequence of interest are “operably linked” when the sequences are functionally connected so as to permit transcription of the sequence of interest to be mediated or modulated by the transcriptional regulatory region. In some embodiments, to be operably linked, a transcriptional regulatory region may be located on the same strand as the sequence of interest. The transcriptional regulatory region may in some embodiments be located 5′ of the sequence of interest. In such embodiments, the transcriptional regulatory region may be directly 5′ of the sequence of interest or there may be intervening sequences between these regions. Transcriptional regulatory sequences may in some embodiments be located 3′ of the sequence of interest. The operable linkage of the transcriptional regulatory region and the sequence of interest may require appropriate molecules (such as transcriptional activator proteins) to be bound to the transcriptional regulatory region, the invention therefore encompasses embodiments in which such molecules are provided, either in vitro or in vivo.

The sequence of DNA that is transcribed by RNA polymerase into messenger RNA generally begins with a sequence that is not translated into protein, referred to as a 5′ non-translated end of a strand of mRNA, that may attach to a ribosome. This non-translated sequenced (CAP) may be added after transcription of the gene. The mRNA moves through the ribosome until a “start codon” is encountered. The start codon is usually the series of three bases, AUG; rarely, the codon GUG may cause the initiation of translation.

The next sequence of bases in a gene is usually called the coding sequence or the structural sequence. The start codon directs the ribosome to begin connecting a series of amino acids to each other by peptide bonds to form a polypeptide, starting with methionine, which forms the amino terminal end of the polypeptide (the methionine residue may be subsequently removed from the polypeptide by other enzymes). The bases which follow the AUG start codon are divided into sets of 3, each of which is a codon. The “reading frame,” which specifies how the bases are grouped together into sets of 3, is determined by the start codon. Each codon codes for the addition of a specific amino acid to the polypeptide being formed. Three of the codons (UAA, UAG, and UGA) are typically “stop” codons; when a stop codon reaches the translation mechanism of a ribosome, the polypeptide that was being formed disengages from the ribosome, and the last preceding amino acid residue becomes the carboxyl terminal end of the polypeptide.

The region of mRNA which is located on the 3′ side of a stop codon in a monocistronic gene is referred to as a 3′ non-translated region. This region may be involved in the processing, stability, and/or transport of the mRNA after it is transcribed. This region may also include a polyadenylation signal which is recognized by an enzyme in the cell that adds a substantial number of adenosine residues to the mRNA molecule, to form a poly-A tail.

Various genes and nucleic acid sequences of the invention may be recombinant sequences. The term “recombinant” means that something has been recombined, so that with reference to a nucleic acid construct the term refers to a molecule that is comprised of nucleic acid sequences that have at some point been joined together or produced by means of molecular biological techniques. The term “recombinant” when made with reference to a protein or a polypeptide refers to a protein or polypeptide molecule which is expressed using a recombinant nucleic acid construct created by means of molecular biological techniques. The term “recombinant” when made in reference to genetic composition refers to a gamete or progeny or cell or genome with new combinations of alleles that did not occur in the naturally-occurring parental genomes. Recombinant nucleic acid constructs may include a nucleotide sequence which is ligated to, or is manipulated to become ligated to, a nucleic acid sequence to which it is not ligated in nature, or to which it is ligated at a different location in nature. Referring to a nucleic acid construct as “recombinant” therefore indicates that the nucleic acid molecule has been manipulated by human intervention using genetic engineering.

Recombinant nucleic acid constructs may for example be introduced into a host cell by transformation. Such recombinant nucleic acid constructs may include sequences derived from the same host cell species or from different host cell species, which have been isolated and reintroduced into cells of the host species.

Recombinant nucleic acid sequences may become integrated into a host cell genome, either as a result of the original transformation of the host cells, or as the result of subsequent recombination and/or repair events. Alternatively, recombinant sequences may be maintained as extra-chromosomal elements. Such sequences may be reproduced, for example by using an organism such as a transformed yeast strain as a starting strain for strain improvement procedures implemented by mutation, mass mating or protoplast fusion. The resulting strains that preserve the recombinant sequence of the invention are themselves considered “recombinant” as that term is used herein.

In various aspects of the invention, nucleic acid molecules may be chemically synthesized using techniques such as are disclosed, for example, in Itakura et al. U.S. Pat. No. 4,598,049; Caruthers et al. U.S. Pat. No. 4,458,066; and Itakura U.S. Pat. Nos. 4,401,796 and 4,373,071. Such synthetic nucleic acids are by their nature “recombinant” as that term is used herein (being the product of successive steps of combining the constituent parts of the molecule).

Transformation is the process by which the genetic material carried by a cell is altered by incorporation of one or more exogenous nucleic acids into the cell. For example, yeast may be transformed using a variety of protocols (Gietz et al., 1995). Such transformation may occur by incorporation of the exogenous nucleic acid into the genetic material of the cell, or by virtue of an alteration in the endogenous genetic material of the cell that results from exposure of the cell to the exogenous nucleic acid. Transformants or transformed cells are cells, or descendants of cells, that have been genetically altered through the uptake of an exogenous nucleic acid. As these terms are used herein, they apply to descendants of transformed cells where the desired genetic alteration has been preserved through subsequent cellular generations, irrespective of other mutations or alterations that may also be present in the cells of the subsequent generations.

In alternative aspects, the invention relates to yeast strains used in fermentation to produce a variety of products, such as wine, beer, dough, ethanol or vinegar. In alternative embodiments, the invention may for example utilize S. cerevisiae yeast strains, S. bayanus yeast strains, or Schizosaccharomyces yeast strains. Transformed host cells for use in wine-making may for example include strains of S. cerevisiae or Schizosaccharomyces, such as Bourgovin (RC 212 Saccharomyces cerevisiae), ICV D-47 Saccharamyces cerevisiae, 71B-1122 Saccharomyces cerevisiae, K1V-1116 Saccharomyces cerevisiae, EC-1118 Saccharomyces bayanus, Vin13, Vin7, N96, and WE352. There are a variety of commercial sources for yeast strains, such as Lallemand Inc. of Montreal Quebec, Canada.

In some embodiments, aspects of the invention may make use of endogenous or heterologous enzymes having urea degrading activity, such as the urea carboxylase and allophanate hydrolase activity of DUR1,2. These enzymes may for example be homologous to DUR1,2 or to regions of DUR1,2 having the relevant activity.

The degree of homology between sequences (such as native DUR1,2 protein or native DUR1,2 nucleic acid sequences and the sequence of an alternative protein or nucleic acid for use in the invention) may be expressed as a percentage of identity when the sequences are optimally aligned, meaning the occurrence of exact matches between the sequences. Optimal alignment of sequences for comparisons of identity may be conducted using a variety of algorithms, such as the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math 2: 482, the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, the search for similarity method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85: 2444, and the computerised implementations of these algorithms (such as GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, Wis., U.S.A.). Sequence alignment may also be carried out using the BLAST algorithm, described in Altschul et al., 1990, J. Mol. Biol. 215:403-10 (using the published default settings). Software for performing BLAST analysis may be available through the National Center for Biotechnology Information (through the internet at http://www.ncbi.nlm.nih.gov/). The BLAST algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold. Initial neighbourhood word hits act as seeds for initiating searches to find longer HSPs. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction is halted when the following parameters are met: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST programs may use as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (Henikoff and Henikoff, 1992, Proc. Natl. Acad. Sci. USA 89: 10915-10919) alignments (B) of 50, expectation (E) of 10 (which may be changed in alternative embodiments to 1 or 0.1 or 0.01 or 0.001 or 0.0001; although E values much higher than 0.1 may not identify functionally similar sequences, it is useful to examine hits with lower significance, E values between 0.1 and 10, for short regions of similarity), M=5, N=4, for nucleic acids a comparison of both strands. For protein comparisons, BLASTP may be used with defaults as follows: G=11 (cost to open a gap); E=1 (cost to extend a gap); E=10 (expectation value, at this setting, 10 hits with scores equal to or better than the defined alignment score, S, are expected to occur by chance in a database of the same size as the one being searched; the E value can be increased or decreased to alter the stringency of the search.); and W=3 (word size, default is 11 for BLASTN, 3 for other blast programs). The BLOSUM matrix assigns a probability score for each position in an alignment that is based on the frequency with which that substitution is known to occur among consensus blocks within related proteins. The BLOSUM62 (gap existence cost=11; per residue gap cost=1; lambda ratio=0.85) substitution matrix is used by default in BLAST 2.0. A variety of other matrices may be used as alternatives to BLOSUM62, including: PAM30 (9,1,0.87); PAM70 (10,1,0.87) BLOSUM80 (10,1,0.87); BLOSUM62 (11,1,0.82) and BLOSUM45 (14,2,0.87). One measure of the statistical similarity between two sequences using the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. In alternative embodiments of the invention, nucleotide or amino acid sequences are considered substantially identical if the smallest sum probability in a comparison of the test sequences is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

Nucleic acid sequences of the invention may in some embodiments be substantially identical, such as substantially identical to DUR1,2 protein or DUR1,2 nucleic acid sequences. The substantial identity of such sequences may be reflected in percentage of identity when optimally aligned that may for example be greater than 50%, 80% to 100%, at least 80%, at least 90% or at least 95%, which in the case of gene targeting substrates may refer to the identity of a portion of the gene targeting substrate with a portion of the target sequence, wherein the degree of identity may facilitate homologous pairing and recombination and/or repair. An alternative indication that two nucleic acid sequences are substantially identical is that the two sequences hybridize to each other under moderately stringent, or preferably stringent, conditions. Hybridization to filter-bound sequences under moderately stringent conditions may, for example, be performed in 0.5 M NaHPO₄, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.2×SSC/0.1% SDS at 42° C. (see Ausubel, et al. (eds), 1989, Current Protocols in MolecularBiology, Vol. 1, Green Publishing Associates, Inc., and John Wiley & Sons, Inc., New York, at p. 2.10.3). Alternatively, hybridization to filter-bound sequences under stringent conditions may, for example, be performed in 0.5 M NaHPO₄, 7% SDS, 1 mM EDTA at 65° C., and washing in 0.1×SSC/0.1% SDS at 68° C. (see Ausubel, et al. (eds), 1989, supra). Hybridization conditions may be modified in accordance with known methods depending on the sequence of interest (see Tijssen, 1993, Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2 “Overview of principles of hybridization and the strategy of nucleic acid probe assays”, Elsevier, New York). Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point for the specific sequence at a defined ionic strength and pH. Washes for stingent hybridization may for example be of at least 15 minutes, 30 minutes, 45 minutes, 60 minutes, 75 minutes, 90 minutes, 105 minutes or 120 minutes.

It is well known in the art that some modifications and changes can be made in the structure of a polypeptide, such as DUR1,2, without substantially altering the biological function of that peptide, to obtain a biologically equivalent polypeptide. In one aspect of the invention, proteins, urea carboxylase and/or allophanate hydrolase activity may include proteins that differ from the native DUR1,2 sequence by conservative amino acid substitutions. As used herein, the term “conserved amino acid substitutions” refers to the substitution of one amino acid for another at a given location in the protein, where the substitution can be made without substantial loss of the relevant function. In making such changes, substitutions of like amino acid residues can be made on the basis of relative similarity of side-chain substituents, for example, their size, charge, hydrophobicity, hydrophilicity, and the like, and such substitutions may be assayed for their effect on the function of the protein by routine testing.

In some embodiments, conserved amino acid substitutions may be made where an amino acid residue is substituted for another having a similar hydrophilicity value (e.g., within a value of plus or minus 2.0), where the following may be an amino acid having a hydropathic index of about −1.6 such as Tyr (−1.3) or Pro (−1.6)s are assigned to amino acid residues (as detailed in U.S. Pat. No. 4,554,101, incorporated herein by reference): Arg (+3.0); Lys (+3.0); Asp (+3.0); Glu (+3.0); Ser (+0.3); Asn (+0.2); Gln (+0.2); Gly (0); Pro (−0.5); Thr (−0.4); Ala (−0.5); His (−0.5); Cys (−1.0); Met (−1.3); Val (−1.5); Leu (−1.8); lle (−1.8); Tyr (−2.3); Phe (−2.5); and Trp (−3.4).

In alternative embodiments, conserved amino acid substitutions may be made where an amino acid residue is substituted for another having a similar hydropathic index (e.g., within a value of plus or minus 2.0). In such embodiments, each amino acid residue may be assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics, as follows: lle (+4.5); Val (+4.2); Leu (+3.8); Phe (+2.8); Cys (+2.5); Met (+1.9); Ala (+1.8); Gly (−0.4); Thr (−0.7); Ser (−0.8); Trp (−0.9); Tyr (−1.3); Pro (−1.6); His (−3.2); Glu (−3.5); Gln (−3.5); Asp (−3.5); Asn (−3.5); Lys (−3.9); and Arg (−4.5).

In alternative embodiments, conserved amino acid substitutions may be made where an amino acid residue is substituted for another in the same class, where the amino acids are divided into non-polar, acidic, basic and neutral classes, as follows: non-polar: Ala, Val, Leu, Ile, Phe, Trp, Pro, Met; acidic: Asp, Glu; basic: Lys, Arg, His; neutral: Gly, Ser, Thr, Cys, Asn, Gin, Tyr.

In alternative embodiments, conservative amino acid changes include changes based on considerations of hydrophilicity or hydrophobicity, size or volume, or charge. Amino acids can be generally characterized as hydrophobic or hydrophilic, depending primarily on the properties of the amino acid side chain. A hydrophobic amino acid exhibits a hydrophobicity of greater than zero, and a hydrophilic amino acid exhibits a hydrophilicity of less than zero, based on the normalized consensus hydrophobicity scale of Eisenberg et al. (J. Mol. Bio. 179:125-142, 184). Genetically encoded hydrophobic amino acids include Gly, Ala, Phe, Val, Leu, lie, Pro, Met and Trp, and genetically encoded hydrophilic amino acids include Thr, His, Glu, Gln, Asp, Arg, Ser, and Lys. Non-genetically encoded hydrophobic amino acids include t-butylalanine, while non-genetically encoded hydrophilic amino acids include citrulline and homocysteine.

Hydrophobic or hydrophilic amino acids can be further subdivided based on the characteristics of their side chains. For example, an aromatic amino acid is a hydrophobic amino acid with a side chain containing at least one aromatic or heteroaromatic ring, which may contain one or more substituents such as —OH, —SH, —CN, —F, —Cl, —Br, —I, —NO₂, —NO, —NH₂, —NHR, —NRR, —C(O)R, —C(O)OH, —C(O)OR, —C(O)NH₂, —C(O)NHR, —C(O)NRR, etc., where R is independently (C₁-C₆) alkyl, substituted (C₁-C₆) alkyl, (C₁-C₆) alkenyl, substituted (C₁-C₆) alkenyl, (C₁-C₆) alkynyl, substituted (C₁-C₆) alkynyl, (C₅-C₂₀) aryl, substituted (C₅-C₂₀) aryl, (C₆-C₂₆) alkaryl, substituted (C₆-C₂₆) alkaryl, 5-20 membered heteroaryl, substituted 5-20 membered heteroaryl, 6-26 membered alkheteroaryl or substituted 6-26 membered alkheteroaryl. Genetically encoded aromatic amino acids include Phe, Tyr, and Tryp.

An apolar amino acid is a hydrophobic amino acid with a side chain that is uncharged at physiological pH and which has bonds in which a pair of electrons shared in common by two atoms is generally held equally by each of the two atoms (i.e., the side chain is not polar). Genetically encoded apolar amino acids include Gly, Leu, Val, Ile, Ala, and Met. Apolar amino acids can be further subdivided to include aliphatic amino acids, which is a hydrophobic amino acid having an aliphatic hydrocarbon side chain. Genetically encoded aliphatic amino acids include Ala, Leu, Val, and Ile.

A polar amino acid is a hydrophilic amino acid with a side chain that is uncharged at physiological pH, but which has one bond in which the pair of electrons shared in common by two atoms is held more closely by one of the atoms. Genetically encoded polar amino acids include Ser, Thr, Asn, and Gin.

An acidic amino acid is a hydrophilic amino acid with a side chain pKa value of less than 7. Acidic amino acids typically have negatively charged side chains at physiological pH due to loss of a hydrogen ion. Genetically encoded acidic amino acids include Asp and Glu. A basic amino acid is a hydrophilic amino acid with a side chain pKa value of greater than 7. Basic amino acids typically have positively charged side chains at physiological pH due to association with hydronium ion. Genetically encoded basic amino acids include Arg, Lys, and His.

It will be appreciated by one skilled in the art that the above classifications are not absolute and that an amino acid may be classified in more than one category. In addition, amino acids can be classified based on known behaviour and or characteristic chemical, physical, or biological properties based on specified assays or as compared with previously identified amino acids.

In various aspects of the invention, the urea degrading activity of a host may be adjusted so that it is at a desired level under fermentation conditions, such as under wine fermentation conditions. The term “fermentation conditions” or “fermenting conditions” means conditions under which an organism, such as S. cerevisiae, produces energy by fermentation, i.e. culture conditions under which fermentation takes place. Broadly defined, fermentation is the sum of anaerobic reactions that can provide energy for the growth of microorganisms in the absence of oxygen. Energy in fermentation is provided by substrate-level phosphorylation. In fermentation, an organic compound (the energy source) serves as a donor of electrons and another organic compound is the electron acceptor. Various organic substrates may be used for fermentation, such as carbohydrates, amino acids, purines and pyrimidines. In one aspect, the invention relates to organisms, such as yeast, capable of carbohydrate fermentation to produce ethyl alcohol.

In wine fermentation, the culture conditions of the must are derived from the fruit juice used as starting material. For example, the main constituents of grape juice are glucose (typically about 75 to 150 g/l), fructose (typically about 75 to 150 gA), tartaric acid (typically about 2 to 10 g/l), malic acid (typically about 1 to 8 g/l) and free amino acids (typically about 0.2 to 2.5 g/l). However, virtually any fruit or sugary plant sap can be processed into an alcoholic beverage in a process in which the main reaction is the conversion of a carbohydrate to ethyl alcohol.

Wine yeast typically grows and ferments in a pH range of about 4 to 4.5 and requires a minimum water activity of about 0.85 (or a relative humidity of about 88%). The fermentation may be allowed to proceed spontaneously, or can be started by inoculation with a must that has been previously fermented, in which case the juice may be inoculated with populations of yeast of about 10⁶ to about 10⁷ cfu/ml juice. The must may be aerated to build up the yeast population. Once fermentation begins, the rapid production of carbon dioxide generally maintains anaerobic conditions. The temperature of fermentation is usually from 10° C. to 30° C., and the duration of the fermentation process may for example extend from a few days to a few weeks.

In one aspect, the present invention provides yeast strains that are capable of reducing the concentration of ethyl carbamate in fermented alcoholic beverages. For example, the invention may be used to provide wines having an ethyl carbamate concentration of less than 40 ppb (μg/L), 35 ppb, 30 ppb, 25 ppb, 20 ppb, 15 ppb, 10 ppb or 5 ppb (or any integer value between 50 ppb and 1 ppb). In alternative embodiments, the invention may be used to provide fortified wines or distilled spirits having an ethyl carbamate concentration of less than about 500 ppb, 400 ppb, 300 ppb, 200 ppb, 150 ppb, 100 ppb, 90 ppb, 80 ppb, 70 ppb, 60 ppb, 50 ppb, 40 ppb, 30 ppb, 20 ppb or 10 ppb (or any integer value between 500 ppb and 10 ppb).

In alternative embodiments, the invention may provide yeast strains that are capable of maintaining a reduced urea concentration in grape musts. For example, urea concentrations may be maintained below about 15 mg/l, 10 mg/l, 5 mg/l, 4 mg/l, 3 mg/l, 2 mg/l or 1 mg/l.

In one aspect, the invention provides methods for selecting natural mutants of a fermenting organism having a desired level of urea degrading activity under fermenting conditions. For example, yeast strains may be selected that lacking NCR of DUR1,2. For an example of mutation and selection protocols for yeast, see U.S. Pat. No. 6,140,108 issued to Mortimer et al. Oct. 31, 2000. In such methods, a yeast strain may be treated with a mutagen, such as ethylmethane sulfonate, nitrous acid, or hydroxylamine, which produce mutants with base-pair substitutions. Mutants with altered urea degrading activity may be screened for example by plating on an appropriate medium.

In alternative embodiments, site directed mutagenesis may be employed to alter the level of urea degrading activity in a host. For example, site directed mutagenesis may be employed to remove NCR mediating elements from the DUR1,2 promoter. For example, the GATAA(G) boxes in the native DUR1,2 promoter sequence, as shown in FIG. 2, may be deleted or modified by substitution. In one embodiment, for example, one or both of the GATAA boxes may be modified by substituting a T for the G, so that the sequence becomes TATAA. Methods of site directed mutagenesis are for example disclosed in: Rothstein, 1991; Simon and Moore, 1987; Winzeler et al., 1999; and, Negrittoet al., 1997. In alternative embodiments, the genes encoding for Gln3p and Gat1p that mediate NCR in S. cerevisiae may also be mutated to modulate NCR.

The relative urea degrading enzymatic activity of a yeast strain of the invention may be measured relative to an untransformed parent strain. For example, transformed yeast strains of the invention may be selected to have greater urea degrading activity than a parent strain under fermenting conditions, or an activity that is some greater proportion of the parent strain activity under the same fermenting conditions, such as at least 150%, 200%, 250%, 300%, 400% or 500% of the parent strain activity. Similarly, the activity of enzymes expressed or encoded by recombinant nucleic acids of the invention may be determined relative to the non-recombinant sequences from which they are derived, using similar multiples of activity.

In one aspect of the invention, a vector may be provided comprising a recombinant nucleic acid molecule having the DUR1,2 coding sequence, or homologues thereof, under the control of a heterologous promoter sequence that mediates regulated expression of the DUR1,2 polypeptide. To provide such vectors, the DUR1,2 open reading frame (ORF) from S. cerevisiae may be inserted into a plasmid containing an expression cassette that will regulate expression of the recombinant DUR1,2 gene. The recombinant molecule may be introduced into a selected yeast strain to provide a transformed strain having altered urea degrading activity. In alternative embodiments, expression of a native DUR1,2 coding sequence homologue in a host such as S. cerevisiae may also be effected by replacing the native promoter with another promoter. Additional regulatory elements may also be used to construct recombinant expression cassettes utilizing an endogenous coding sequence. Recombinant genes or expression cassettes may be integrated into the chromosomal DNA of a host such as S. cerevisiae.

Promoters for use in alternative aspects of the invention may be selected from suitable native S. cerevisiae promoters, such as the PGK1 or CAR1 promoters. Such promoters may be used with additional regulator elements, such as the PGK1 and CAR1. terminators. A variety of native or recombinant promoters may be used, where the promoters are selected or constructed to mediate expression of urea degrading activities, such as DUR1,2 activities, under selected conditions, such as wine making conditions.

According to one aspect of the invention, a method of fermenting a carbohydrate is provided, such as a method of fermenting wine, using a host, such as a yeast strain, transformed with a recombinant nucleic acid that modulates the urea degrading activity of the host. For example, the NCR of the DUR1,2 gene may be modulated to enhance the degradation of urea to ammonia and carbon dioxide in a wine making yeast strain. In accordance with this aspect of the invention, fermentation of a grape must with the yeast strain may be carried out so as to result in the production of limited amounts of ethyl carbamate.

In one embodiment, using standard recombinant methods (Ausubel et al., 1995) a phleomycin resistance gene cassette was cloned into a yeast shuttle vector, to produce a plasmid called pJC2phleo. The DUR1,2 gene open reading frame (ORF) was amplified by PCR and cloned into the pJC2phleo plasmid, to produce a vector called pJC2/DUR1,2phleo. The pJC2/DUR1,2phleo vector is a multicopy, episomal S. cerevisiae-E. coli shuttle plasmid in which the DUR1,2 coding sequence is inserted between the regulatory sequences of the yeast phosphoglycerate kinase (PGK1) gene, so that the PGK1 promoter and terminator sequences are operatively linked to the DUR1,2 coding sequence. A LEU2 marker allows the selection of transformed yeast cells that are auxotrophic for leucine. The pJC2/DUR1,2phleo plasmid also contains the Tn5Ble gene driven by the constitutive TEF1 yeast promoter and CYC1 terminator. In vivo analysis of transformants showed that the urea degradation capacity of S. cerevisiae cells transformed with the pJC/DUR1,2phleo plasmid was significantly increased, compared to cells transformed with pJC2phleo, as measured by the concentration of urea in the culture media.

Although various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in this art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. Numeric ranges are inclusive of the numbers defining the range. In the specification, the word “comprising” is used as an open-ended term, substantially equivalent to the phrase “including, but not limited to”, and the word “comprises” has a corresponding meaning. Citation of references herein shall not be construed as an admission that such references are prior art to the present invention. All publications, including but not limited to patents and patent applications, cited in this specification are incorporated herein by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein and as though fully set forth herein. The invention includes all embodiments and variations substantially as hereinbefore described and with reference to the examples and drawings.

Entry of the sequence listing is directed into this application. The Sequence Listing material on the compact disc containing file name “80021-398.seq.07.jan.2003.v1.txt,”byte size 4 KB and created on Jan. 7, 2003, is hereby incorporated-by-refernce.

References

The following documents are hereby incorporated by reference:

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Ausubel, F M, Brent, R, Kingston, R E, Moore, D. D., Seidman, J G, Smith, J A, Struhl, K eds. Current Protocols in Molecular Biology. 1987-2000. John Wiley and Sons, Inc.

Dietmaier, W. and Pabry S. (1995). Protocol: DI/tri nucleotide Sticky End Cloning. Boerhinger Mannheim PCR Application Manual.

Genbauffe, F. S., and Cooper, T. G. 1991. The urea amidolyase (DUR1,2) gene of Saccharomyces cerevisiae. DNA Seq. 2(1): 19-32.

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1. A method of making a fermented product comprising maintaining under fermenting conditions a S. cerevisiae yeast strain transformed to express a urea degrading enzyme having urea carboxylase and allophanate hydrolase activity from a gene comprising a constitutively active heterologous yeast promoter, and a coding sequence that is at least 80% identical when optimally aligned to a S. cerevisiae DUR1,2 coding sequence of SEQ ID NO:6, and wherein the urea degrading enzyme comprises a DUR1,2 protein of SEQ ID NO:7, and wherein the fermented product is selected from the group consisting of alcoholic beverages, distilled alcoholic beverages, wines, beers, doughs, ethanol and vinegar.
 2. The method of claim 1, wherein the coding sequence comprises an open reading frame at least 90% identical to the S. cerevisiae DUR1,2 coding sequence of SEQ ID NO:6.
 3. The method of claim 1, wherein the coding sequence comprises an open reading frame at least 95% identical to the S. cerevisiae DUR1,2 coding sequence of SEQ ID NO:6.
 4. The method of claim 1, wherein the coding sequence comprises the S. cerevisiae DUR1,2 coding sequence of SEQ ID NO:6.
 5. The method of claim 2, wherein the coding sequence encodes a DUR1,2 protein of SEQ ID NO:7.
 6. The method of claim 3, wherein the coding sequence encodes a DUR1,2 protein of SEQ ID NO:7.
 7. The method of claim 1, wherein the fermented product is an alcoholic beverage.
 8. The method of claim 2, wherein the fermented product is an alcoholic beverage.
 9. The method of claim 3, wherein the fermented product is an alcoholic beverage.
 10. The method of claim 4, wherein the fermented product is an alcoholic beverage.
 11. The method of claim 1, wherein the fermented product is wine.
 12. The method of claim 11, wherein the wine has an ethyl carbamate concentration of less than 30 ppb. 